We investigated the effects of stent generation on 2-year clinical outcomes between prediabetes and diabetes patients after acute myocardial infarction (AMI). A total of 13,895 AMI patients were classified into normoglycemia (group A: 3673), prediabetes (group B: 5205), and diabetes (group C: 5017). Thereafter, all three groups were further divided into first-generation (1G)-drug-eluting stent (DES) and second-generation (2G)-DES groups. Patient-oriented composite outcomes (POCOs) defined as all-cause death, recurrent myocardial infarction (Re-MI), and any repeat revascularization were the primary outcome. Stent thrombosis (ST) was the secondary outcome. In both prediabetes and diabetes groups, the cumulative incidences of POCOs, any repeat revascularization, and ST were higher in the 1G-DES than that in the 2G-DES. In the diabetes group, all-cause death and cardiac death rates were higher in the 1G-DES than that in the 2G-DES. In both stent generations, the cumulative incidence of POCOs was similar between the prediabetes and diabetes groups. However, in the 2G-DES group, the cumulative incidences of Re-MI and all-cause death or MI were significantly higher in the diabetes group than that in the prediabetes group. To conclude, 2G-DES was more effective than 1G-DES in reducing the primary and secondary outcomes for both prediabetes and diabetes groups. Moreover, almost two thirds of those presenting with CAD have either diabetes or prediabetes2. Coronary vessels in patients with diabetes usually present extensive atherosclerosis with a larger number of significant stenosis, longer lesions, and more diffuse disease4. Therefore, despite advances in interventional skill, devices, and antiplatelet agents, outcomes of coronary revascularization in patients with diabetes have been poorer than those without6. Percutaneous coronary intervention (PCI) in patients with diabetes is associated with increased incidence of restenosis, repeat revascularization, stent thrombosis (ST), and all-cause mortality than those without4. Drug-eluting stents (DES) reduce the risk of restenosis as compared with bare-metal stents (BMS). However, ST remains a major concern after the implantation of first-generation (1G)-DES in patients with diabetes7. Relative superiority between the 1G- and 2G-DESs in patients with diabetes remains controversial11. Although recent reports revealed that prediabetes is an intergrade between normoglycemia and diabetes14, PCI patients with prediabetes were prone to experience adverse clinical events. Individuals with prediabetes are important and common patients who visit interventional cardiologists. However, the main treatment strategies for hyperglycemia are focused on the patients with diabetes rather those with prediabetes15. Moreover, studies regarding the effects of the 1G-DES and 2G-DES on clinical outcomes between prediabetes and diabetes patients after AMI were limited. To better understand the characteristic of prediabetes, we compared the 2-year clinical outcomes of the 1G-DES and 2G-DES under two different glycemic states (prediabetes and diabetes).Diabetes mellitus  is regarded as a “coronary artery disease (CAD) risk equivalent”Table Cumulative incidences of major clinical outcomes during the 2-year follow-up period are summarized in Tables After the adjustment, the cumulative incidences of POCOs , any repeat revascularization , and ST  were significantly higher in the 1G-DES than that in the 2G-DES group.After the adjustment, the cumulative incidences of POCOs , all-cause death , CD , any repeat revascularization , and ST  were significantly higher in the 1G-DES group than that in the 2G-DES group.After the adjustment, the cumulative incidences of POCOs, all-cause death, CD, Re-MI, all-cause death or MI, and any repeat revascularization were similar between the 1G-DES and 2G-DES groups. However, the cumulative incidence of ST  was significantly higher in the 1G-DES than that in the 2G-DES group.Cumulative incidences of POCOs  and ST  were similar between prediabetes and diabetes groups. The cumulative incidence of any repeat revascularization was significantly higher in the prediabetes than that in the normoglycemia group . Cumulative incidences of POCOs  and any repeat revascularization  were significantly higher in the diabetes than that in the normoglycemia group.Cumulative incidences of POCOs  and ST  were similar between prediabetes and diabetes groups. However, cumulative incidences of Re-MI  and all-cause death or MI  in the diabetes group were significantly higher than that in the prediabetes group. Cumulative incidences of POCOs , all-cause death , CD , and all-cause death or MI  were significantly higher in the prediabetes than that in the normoglycemia group. Cumulative incidences of POCOs , all-cause death , CD , Re-MI , all-cause death or MI , any repeat revascularization , and ST  were significantly higher in the diabetes than that in the normoglycemia group.Table The primary findings of this study are as follows: (1) in both prediabetes and diabetes groups, the cumulative incidences of POCOs, any repeat revascularization, and ST were higher in the 1G-DES than that in the 2G-DES; (2) in the diabetes group, the cumulative incidences of all-cause death and CD were higher in the 1G-DES than that in the 2G-DES; (3) in the normoglycemia group, the cumulative incidence of ST was higher in the 1G-DES than that in the 2G-DES; and (4) in two different stent generations, the cumulative incidence of POCOs was similar between the prediabetes and diabetes groups. However, in the 2G-DES group, the cumulative incidences of Re-MI and all-cause death or MI were higher in the diabetes group than that in the prediabetes group.16. Although previous reports demonstrated that the higher rates of repeat revascularizations and mortality after PCI in patients with diabetes are caused by restenosis and disease progression6, comparative clinical outcomes between prediabetes and diabetes were not well illuminated especially, between 1G-DES and 2G-DES. Some recent reports showed that prediabetes is associated with poorer clinical outcomes including cardiovascular mortality and patients with prediabetes and diabetes have similar higher risk profiles compared with normoglycemia17.Hyperglycemia, elevated free fatty acid level, and increased amount of circulating glucosylated serum products can accelerate atherosclerosis and vascular injury in patients with diabetes by inducing endothelial dysfunction and vascular inflammation19, ST remains a major concern after the DES implantation, especially in diabetes4. Relative superiority between the 1G-DES and 2G-DES in patients with AMI and diabetes remains controversial, and most previous studies were not performed during the prediabetes stage21. In our study, the cumulative incidence of POCOs was significantly higher in the 1G-DES than that in the 2G-DES in both prediabetes and diabetes groups. Moreover, in two different stent generations, the cumulative incidence of POCOs was similar between the prediabetes and diabetes groups . Von Birgelen et al.22 reported the results of the BIO-RESORT Silent Diabetes Study. In their study, the cumulative incidence of major adverse cardiac events was different between patients with prediabetes (5.5%) and normoglycemia (3.0%) . As mentioned, despite the combination of new platforms, more biocompatible polymers were utilized in 2G-DES, the relative superiority between 1G- and 2G-DESs in patients with diabetes remains controversial11. In the SPIRIT V Diabetic Study10, everolimus-eluting stent (EES) was superior to paclitaxel-eluting stent (PES) for in-stent late loss at 9 months. The composite death, MI, and TVR rates were the same in the two groups at 1 year. Bavishi et al.9 reported that EES showed significantly lower incidence rates of MACEs by 18% and ST by 46% as compared with the 1G-DES. Moreover, the EES showed a trend toward reduced incidence rates of target lesion revascularization (TLR) and TVR (p = 0.05). In this study, based on the cumulative incidences of POCOs, any revascularization rate was significantly higher in the 1G-DES than that in the 2G-DES group in both prediabetes and diabetes group. Therefore, the major clinical outcomes of our study could reflect the meta-analysis results of Bavishi et al.’s study9.Although DES improved outcomes of high-risk patients by reducing the rate of restenosis as compared with BMSps Table . In a su9, EES reduced the incidence of ST by 46%  as compared with the 1G-DES in patients with diabetes. The cumulative incidence of ST also higher in the 1G-DES than that in the 2G-DES in patients with normoglycemia. Our result is consistent with the result of Nakatsuma et al. study23. This low cumulative incidence of 2G-DES may be related with relatively thin stent struts (50–90 μm) and improved ability for deliverability while maintaining an adequate radial strength24 and more compatible and thromboresistant than those in the 1G-DES25. However, in our study, the occurrence of ST was high within 6 months after index PCI -guided PCI28 could reduce MACE rate, the number of PCI base on these intracoronary image- or functional study-based PCI were less than 30% in our study. Unfortunately, currently under the Korea’s health insurance system, the reimbursement program for the use of IVUS, optical coherence of tomography, or fractional flow reserve during the PCI is very limited or absent29.The overall rate of ST was also higher in the 1G-DES than in the 2G-DES in all three different glycemic groups . With regard to prediabetes, follow-up data on the comparative long-term effects of 1G-DES and 2G-DES implantation were limited. According to Bavishi et al.’s report6, 2G-DES showed decreased incidences of all-cause death  and CD  compared with 1G-DES in diabetes group after adjustment  in their meta-analysis. However, the cumulative incidences of all clinical outcomes were significantly higher in the diabetes than that in the normoglycemia group. These results may reflect hazardous effects of diabetes are sustained even in the era of 2G-DES.Interestingly, comparative clinical outcomes of the two different stent generations according to glycemic status showed some different results Table . Differe30 suggested that the association between diabetes and Re-MI may be related with a direct effect of diabetes. According to recent reports31, the cumulative incidence of Re-MI of the diabetes group was significantly higher than that of the prediabetes group .In our study, in the 1G-DES, the primary and secondary end-points were similar between the prediabetes and diabetes groups. However, in the 2G-DES, the cumulative incidences of Re-MI  and all-cause death or MI  were significantly higher in the diabetes group than that in the prediabetes group. Although the precise mechanisms of the higher incidence of Re-MI in diabetes group are not fully known, one reportMore than 50 high-volume university or community hospitals in South Korea participated in this study. The limited reports on the impact of stent generation on long-term clinical outcomes in AMI patients with prediabetes or diabetes were the motivation for the current study. Thus, we believe that our study may provide significant information to interventional cardiologists who perform PCI in patients with AMI with prediabetes or diabetes.32. Third, the duration and types of antidiabetic treatment are major determinants after PCI in patents with prediabetes or diabetes. However, this study was conducted based on discharge medications, and owing to limitation of registry study, we did not precisely know the adherence or non-adherence of enrolled patients to antidiabetic drugs during the follow-up period. Therefore, this may act as an important bias in this study. Fourth, 2G-DES consisted of durable-polymer-coated DES and biodegradable-polymer-coated DES. The number of biodegradable-polymer DES (BES) was highest in the normoglycemia group (prediabetes: 600/4438 (13.5%); diabetes: 536/4238 (12.6%); normoglycemia: 525/3191 (16.5%); p < 0.001) , and/or RPG ≥ 200 mg/dL (11.1 mmol/L) by repeat testing. If first glycemic status was diabetes and second was prediabetes, or first glycemic status was prediabetes and second was normoglycemia, and this is particularly important in patients with AMI, because such patients reveal hyperglycemia in acute phase. However, in this study, the definitions of prediabetes and diabetes were not based on repeat testing. Moreover, considering the limitation of HbA1c, any other diagnostic tests for diabetes including oral glucose tolerance test are needed for a finer classification. However, detailed information on this variable was not included in the KAMIR. Hence, the results of this study can be altered based on other diagnostic tests and which directly influences the assignment of participants, and this factor may have served as an important bias in this studyIn conclusion, in this study, we observed that 2G-DES was more effective than 1G-DES in reducing POCOs, any repeat revascularization and ST in both prediabetes and diabetes group. Moreover, in two different stent generations, and the cumulative incidence of POCOs was similar between the prediabetes and diabetes groups. However, further studies regarding the most advanced DES technology joined with the most advanced anti-thrombotic regimen are needed to confirm these results.http://www.kamir.or.kr) 33. Among them, patients with incomplete laboratory results , those who were lost to follow-up , those with unidentified blood hemoglobin (Hb) A1c and blood glucose level results , those with different generations of stents were deployed in the same patients , those who received dual antiplatelet therapy (DAPT) less than 12 months , and those who received BMS . Finally, a total of 13,895 patients with AMI who underwent successful implantation were considered for inclusion. Patients were classified into normoglycemia , prediabetes , and diabetes   to achieve an activated clotting time of > 250 s. DAPT (a combination of aspirin 100 mg/day with clopidogrel 75 mg/day or ticagrelor 90 mg twice daily or prasugrel 5–10 mg/day) was recommended for > 12 months for patients who underwent PCI. Triple antiplatelet therapy (TAPT: cilostazol 100 mg twice daily in addition to DAPT) was left to the discretion of the individual operators. Diagnostic coronary angiography and PCI were performed using standard guideline32, prediabetes was defined as an HbA1c of 5.7–6.4% and an FPG of 100–125 mg/dL (5.6–6.9 mmol/L). Diabetes was categorized as either known diabetes defined as ongoing medical treatment for diabetes (insulin or antidiabetics), or newly diagnosed diabetes, defined as an HbA1c level ≥ 6.5%, FPG ≥ 126 mg/dL (7.0 mmol/L), and/or RPG ≥ 200 mg/dL (11.1 mmol/L). If the admission electrocardiogram of patients who complained of chest pain showed ST-segment elevations in at least two contiguous leads of ≥ 2 mm (0.2 mV) in men, or ≥ 1.5 mm (0.15 mV) in women in leads V2–V3 and/or ≥ 1 mm (0.1 mV) in other contiguous chest leads or limb leads or new-onset left bundle branch block, the patients were considered to have STEMI36, whereas patients who did not show persistent ST-segment elevation with increased cardiac biomarkers and with appropriate clinical context were considered to have non-STEMI (NSTEMI)37. In cases of NSTEMI, an early invasive treatment strategy was defined as PCI within 24 h after admission37. A successful PCI was defined as a residual stenosis of < 30% and more than grade 3 flow in Thrombolysis In Myocardial Infarction flow for the infarct-related artery (IRA) after the procedure. The primary outcome of this study was the occurrence of POCOs, defined as all-cause death, Re-MI, or any coronary repeat revascularization38. The secondary outcome was definite or probable ST during the 2-year follow-up period. All-cause death was classified as CD or non-CD. Any repeat revascularization comprised target lesion revascularization, target vessel revascularization, and non-TVR. Re-MI, TLR, TVR, and non-TVR definitions have already been published previously40. The cumulative incidence of ST was defined by the current consensus41.Glycemic status was determined based on medical history and glycated hemoglobin (HbA1c), fasting plasma glucose (FPG), and random plasma glucose (RPG) levels at the index hospitalization. According to the American Diabetes Association clinical practice recommendation14; data were expressed as mean ± standard deviation. For categorical variables, intergroup differences were analyzed using chi-squared test or Fisher’s exact test, as appropriate. Data were expressed as numbers and percentages14. The Kaplan–Meier method was used to estimate various clinical outcomes, and the log-rank test was used to compare intergroup differences . Variables with a p value of < 0.00114 or < 0.0542 in the univariate analysis and conventional risk factors of poor outcomes in the AMI population were considered potential confounding factors and were entered into the multivariate analysis. These included variables shown in Tables 14.For continuous variables, differences between the two groups were evaluated with the unpaired t-test. Additionally, differences among the three glycemic groups were evaluated using analysis of variance or the Jonckheere–Terpstra test, whereas a post-hoc analysis of the two groups was performed using the Hochberg test or Dunnett T3 testSupplementary Information


Nature Communications 10.1038/s41467-021-25967-9, published online 30 September 2021.Correction to: In this article Jingquan Li was incorrectly denoted as being one of the equally contributing authors. The original article has been corrected. Following the publication of the original article, it was noted that, due to a typesetting error, the figure labelling for Figure 6A-G was incorrect in the Results section “EE overcomes PD-L1 based checkpoint blockade resistance”. The PDF and HTML versions of the Article have been corrected.


Dissolution tests were undertaken and controlled in a stirred batch reactor using low-calcium Gamble’s solution and phagolysosomal simulant fluid a surrogate for the lung-lining and macrophage phagolysosomal fluid, respectively. Pre-dispersion in 0.05% BSA-water showed a significant delay or decrease in the dissolution of Al2O3 after testing in both low-calcium Gamble’s solution and PSF. Furthermore, use of the 0.05% BSA pre-dispersion medium influenced the dissolution of ZnO (NM-110) in PSF and ZnO (NM-111) in low-calcium Gamble’s solution and PSF. We hypothesize that BSA forms a protective coating on the particles, which delays or lowers the short-term dissolution of the materials used in this study. Consequently, the type of pre-dispersion medium can affect the results in short-term dissolution testing.Dissolution plays an important role on pulmonary toxicity of nanomaterials (NMs). The influence of contextual parameters on the results from dissolution testing needs to be identified to improve the generation of relevant and comparable data. This study investigated how pre-dispersions made in water, low-calcium Gamble’s solution, phagolysosomal simulant fluid (PSF), and 0.05% bovine serum albumin (BSA) affected the dissolution of the Al Data on the solubility and dissolution rates of manufactured nanomaterials (NMs) and their potential phase transformation in biological systems have received increasing interest during the last few years. The increased interest is not least due to recent demands for this type of information for regulatory read-across and grouping in chemicals registration and risk assessment and for assessing the potential uptake and accumulation in the human body ,2. The sSolubility and dissolution testing can be made using several different procedures and test media. Conceptually, however, there are three approaches  that each have their strengths and weaknesses. To understand and apply the results well and allow mutual acceptance of data, it is necessary to harmonize key conceptual parameters and procedures in the specific test methods as much as possible to allow the generation of comparable and reliable data. Therefore, it is necessary to identify the critical steps and parameters in the protocols applied and understand to what extent they should be defined or controlled.2, and carbon nanotubes were used as model NMs, and the materials were dispersed in water, 2% serum, 0.05% serum albumin in water, 10% bronchoalveolar lavage fluid (in 0.9% NaCl or water), or 0.1% Tween-80 in water [2, no genotoxicity effects were dependent on the dispersion medium used. Conclusively, Hadrup et al. suggested to consider the type of dispersion medium when using intratracheal instillation exposure [2, CeO2, SiO2, and carbon nanotubes, were investigated. The NM dispersions were prepared in porcine lung surfactant and bovine serum albumin and shown to influence the effective dosage after 12 and 24 h. Overall, using bovine serum albumin as a dispersant created stable and narrow size-distribution dispersions; however, porcine lung surfactant resembles more closely the proteins in the lung-lining fluid [In this work, we investigate the potential need to harmonize the pre-dispersion step in a protocol for short-term , dissoln et al. ), and man et al. ,8,9. In n et al. ,10. Prepn et al. . Hadrup n et al. . Carbon in water . The acuexposure . In addiexposure  investigThe quality of the pre-dispersion is critical for achieving reproducible size-analysis (within sub- to micron meter size reflecting the approximate minimum aggregates size) and dosing as well as for enhancement of surface-area-related processes, size-related uptake, and translocation. Relevant for dissolution testing of particles, size and accessible surface area are considered critical physicochemical parameters affecting the dissolution kinetics of NMs ,13. To cA study of the dissolution rates in different test media used for simulating a pulmonary compartment using a continuous flow-through membrane method has clearly shown important differences in the rates and dissolved amounts depending on the media . Consequ2 (NM-104)). These materials have different surface properties and aggregate structures and known differences in solubilities and dissolution rates [2O3-coated TiO2 (NM-104) in two biosimulants as test media using water, test media, and 0.05% w/v bovine serum albumin (BSA) water as pre-dispersion media. The test media were low-calcium Gamble’s solution, a surrogate for the lung-lining fluid [In this study, we studied the role of different pre-dispersion media on dispersibility and dissolution rates using three well-characterized and industrially representative nanomaterials  and organic-inorganic coated TiOon rates ,19,20. Wng fluid , and Phang fluid .2 (NM-104) and ZnO (NM-110 and NM-111) originated from the OECD Working Party on Manufactured Nanomaterials Sponsorship Programme and were all obtained from the Fraunhofer Institute for Molecular Biology and Applied Ecology . The two ZnO materials represent a group of relatively rapid dissolving materials of which ZnO (NM-111) is organically coated with hydrophobic triethoxycaprylsilane, and NM-104 represents a poorly soluble NM coated with Al2O3 and glycerine [TiOlycerine . The key® system, Thermo Fisher Scientific, Waltham, MA, USA), or test media . The 0.05% BSA was prepared using ultrapure water. PSF and low-calcium Gamble’s solution was prepared as described below.The nanomaterials were pre-dispersed at the same acoustic power of 7.35 ± 0.05 Watt following the so-called NANOGENOTOX batch dispersion protocol validated as part of the FP7 NANoREG project . The prew/v 96% ethanol and dispersed with 14.57 mL of solution  to a final concentration of 2.56 mg/mL. A 400 W Branson Sonifier S-450D  equipped with a 13 mm disruptor horn was used to sonicate the particle dispersion for 16 min with a 10% amplitude (approx. 42 W) under constant cooling in an ice-water bath, adding the required acoustic delivered power. Ethanol pre-wetting and the same sonication conditions were applied for all pre-dispersion media to allow dispersion of both hydrophilic (NM-104 and NM-110) and hydrophobic (NM-111) test materials. It should be noted that all results concerning pre-dispersion in 0.05% BSA water as dispersion medium were obtained from Holmfred et al. [Dispersions were prepared by weighing 37.5 mg of NM into a 15 mL glass vial. Powders were pre-wetted with 0.5% ave), zeta potential (Zpot), and polydispersity index (PDI). The instrument was equipped with a 633 nm laser measuring at 173° (non-invasive backscattering). Immediately after preparation of the dispersion, 700 µL was transferred to a disposable folded capillary cell . Prior to analysis, the instrument was equilibrated for 5 min. All samples were analyzed using automatic optimization mode of measurement of configuration, at 25 °C using the viscosity of water (0.8872 cP), and optical parameters of water. Zave and PDI were reported as an average of ten repeated measurements. The Zpot was measured using the Smoluchowski model calculated in automatic mode based on ten repeated measurements. The obtained average Zave values were compared with scanning electron microscopy aggregate sizes and Zave benchmark sizes generated by DLS to qualify dispersions made using the NANOGENOTOX dispersion protocol [The quality and stability of the particle dispersions were evaluated using a Malvern Zetasizer Nano ZS  measuring the hydrodynamic size distribution (Z2 (NM-104) and ZnO (NM-110 and NM-111) was quantified from ELISA reader analysis of total protein concentration in the 0.05% BSA batch dispersion medium after sonication with each of the three test materials using the PierceTM BCA Protein Assay Kit . A total of 1 mL dispersion medium was sampled and added to an Eppendorf tube and centrifuged for 30 min at 20,000 RCF to settle the particles and avoid interference in the ELISA reader. The particle-free supernatant was then sampled, allowed to react with the reagent from the kit in 96-well plates, and analyzed against a BSA calibration curve in the ELISA fluorimeter. All ELISA determinations were based on double tests, and concentrations were derived from BSA calibration curves. Concentrations measured in samples of particle-free dispersion medium were used for reference. The amount of adsorbed BSA was calculated by ascribing the difference between the BSA concentrations in the dispersions with and without sample to adsorption by the particles. Data are given as the BSA-load on the particles (µg BSA/mg particle) by dividing the measured BSA concentration difference by the batch particle concentration of 2.56 mg/mL and the percent adsorbed of added BSA.The amount of adsorbed BSA to the TiOPhagolysosomal simulant fluid (PSF) and low-calcium Gamble’s solution were prepared by dissolving the components listed in 2 and CO2 flow are set to 144 and 5.62 mL/min, respectively. Before testing, low-calcium Gamble’s solution and PSF were adjusted with 0.1 M NaOH or HCl to exactly pH 7.4 and 4.5, respectively, and kept constant throughout testing. The particle suspension was transported gently from the probe sonicator, and 4 mL of suspension was added to each of the test reactors at the start of the test resulting in a nominal NM concentration of 102.4 mg/L. To the reference reactor, 4 mL of the respective pre-dispersion medium was added. At t0, t1, t2, t4, and t24 hours, 4 mL sample was collected using a 5 mL plastic syringe with a steel needle and transferred to 3 kDa Amicon Ultra-4 centrifugal filters  and immediately centrifuged for 30 min at 4000× g, 4400 rpm using an RF+ Sorvall centrifuge . The time from finalising the pre-dispersion and to the t0 h samples were in the centrifuge was approximately 2 min. After centrifugation, the filters were removed, and 0.5 mL of 2% nitric acid was added for stabilization of the dissolved ionic fraction.Dissolution testing was completed in an atmosphere–temperature–pH-controlled (ATempH) stirred batch reactor (SBR) system described and validated in Holmfred et al. . In brie3 and measured using inductively coupled plasma-mass spectrometry (ICP-MS), as specified in 0, t1, t2, t4, and t24 using a Thermo iCAP SQ ICP-MS  equipped with an ASX-560 autosampler . The ICP-MS was further equipped with a Thermo Fisher Scientific quarts cyclonic spray chamber and a PFA-ST MicroFlow nebulizer. During analyses, the plasma power was 1550 W, the plasma gas flow was 14.00 L/min, and the dwell time was set to 100 ms. ICP-MS parameters can be found in The dissolved ionic fraction was diluted 4–1000 times with 2% HNO2 (NM-104) in PSF, the total ionic fraction was quantified against an external calibration curve prepared in 10 times diluted PSF having the same dilution factor as the analyzed samples. ICP-MS certified stock standard solutions 1000 mg/L  with trace elements ≤ 1 µg/L were used to prepare all external calibration curves. The internal standards were likewise prepared in 2% nitric acid from ICP-MS standards (100 mg/L) and diluted to a final concentration of 20 µg/L and added on-line to the sample flow using a T-piece. Blanks and spiked samples were included in all analyzes for quality control of the ICP-MS. The concentrations determined in the reference reactor were subtracted from the measured concentrations of the samples. To avoid carry-over, a rinsing procedure with 2% nitric acid was performed between all sample injections. The limit of detection (LOD) of the measured element in the filtrated sample was calculated using Equation (1)SD is the standard deviation of the concentration of a minimum of five blank samples, and DF is the dilution factor.The 3 kDa filtrates were diluted to an acidic concentration of 2% nitric acid and were quantified against an external calibration curve prepared in 2% nitric acid. Considering the dissolution of TiO0, t1, t2, t4, and t24 hours), the measured dissolved ionic fractions were multiplied with the total dilution factor, corrected for moisture content, coating, and impurities  is the concentration as the function of time, *t is the adjusted time, and Previous studies have described that the dissolution of most NMs follow first-order kinetics ,28,29. Hsamplingt was slightly different for each sample (few seconds) as the samples were collected manually. The centrifuge is kept running for 30 min to ensure that all possible liquid is removed from the filter membrane. However, >75% of the liquid is filtered through within <10 min, according to technical information from Amicon. Using the adjusted time-points, initial, intermediate, and last points were determined according to Fogler (1999) [AC is the concentrationThe 25 min time adjustment was made as an approximation of the total effective delay related to sonication, dosing, sampling, and effective filtration time. The t = 0, SSA is the specific surface area measured by the Brunauer–Emmett–Teller method.The plot of the numerical points as a function of t* were used to determine the initial dissolution rate projected at 0, t1, t2, t4, and t24 hours). p-value ≤ 0.05 was considered significant. Measurements at different time points within a replication were treated as repeated measurements with a first-order autoregressive correlation structure and a model-based covariance matrix. The analyses were conducted by use of the mixed procedure in SAS version 9.4 statistical software .Two-way repeated measures ANOVA  was used to test for independence between dissolution rates and type of pre-dispersion medium. The independence was tested across all measured concentrations (at time points t2 (NM-104) and ZnO (NM-110 and NM-111) are listed in 2 (NM-104). Slightly lower levels of adsorption were found for the triethoxycaprylsilane coated ZnO (NM-111), probably as the organic coating reduces the binding to BSA. The difference in BSA-adsorption observed for ZnO (NM-110 and NM-111) is in agreement with the listed results obtained for the NANOGENOTOX dispersion protocol in Da Silva et al. (2019a) [2 (NM-104) and other TiO2 nanomaterials using a tailored BSA-enhanced dispersion protocol [The results from analysis of BSA adsorption onto TiO 2019a) . Previoua . Previpot measurements on the pre-dispersions of TiO2 (NM-104) and ZnO (NM-110 and NM-111) prepared in water, 0.05% BSA, PSF, or low-calcium Gamble’s solution made with the three test materials in water, 0.05% BSA, PSF, and low-calcium Gamble’s solution before dissolution testing in either PSF or low-calcium Gamble’s solution. The individual DLS size distribution spectra are given in the ave), while dispersion directly in test media generally results in larger Zave values. Only TiO2 (NM-104) disperses better in water than in 0.05% BSA, which is known to be an effect of increased agglomeration in the presence of BSA [pot > −30 mV) [pot of ZnO (NM-110) after pre-dispersion in water (24.6 ± 0.6 and 30.7 mV ± 0.6) indicates that a stable suspension is formed. Water could be considered a dispersion medium for this material in a case-to-case based dispersion medium decision.e of BSA . In gene −30 mV) , and the −30 mV) . Howeverave values were compared to particle average agglomeration sizes (Daggr) determined by scanning electron microscopy and benchmark DLS sizes  for the NANOGENOTOX dispersion protocol  in PSF) times larger Zave sizes than observed in the benchmark DLS data. Good comparability of the dispersion quality parameters listed in 2 (NM-104) used for dissolution testing with low-calcium Gamble’s solution was coarser (ratio = 3.1) compared to all other 0.05% BSA dispersions (ratio 1.1 to 1.6).The level of agglomeration in the different pre-dispersion media was evident when the measured Zn et al. ; Mejia en et al.  and avain et al. ) (Table 2 (NM-104), ZnO (NM-110), and ZnO (NM-111) in low-calcium Gamble’s solution (A) and PSF (B) for the three pre-dispersion media, respectively. p-values of the statistical analyses are presented in 2 (NM-104), the Al2O3 coating dissolved gradually over the 24 h test period, while TiO2 was not dissolved to concentrations above LOD in any of the tests performed in low-calcium Gamble’s solution and PSF  pre-dispersed following the NANOGENOTOX protocol [2O3 coating on TiO2 (NM-103 and NM-104) was observed to dissolve in low-calcium Gamble’s solution and for TiO2 (NM-104) reaching 413 µg/L of Al after 24 h (corresponding to 0.780 mg/L Al2O3 dissolved), comparable to the findings in this study.For TiO and PSF . The dis and PSF . TiO2 isprotocol . In the 2 (NM-104) was highly affected by the pre-dispersion medium, where water and 0.05% BSA formed dispersions with the lowest Zave while the particles agglomerated heavily when dispersed in the test media. However, the samples pre-dispersed in water and test media  showed comparable dissolution profiles  with a significantly 1.2–1.3 fold higher amount of total dissolved Al2O3 after 24 h than after pre-dispersion in 0.05% BSA . A total of 131.8 ± 22.0 µg BSA/mg was found to adsorb to TiO2 (NM-104)  and acidic pH (PSF).We observed that the hydrodynamic size of TiO.05% BSA A,B. The (NM-104) ; the prop-values in Testing of ZnO (NM-110) dissolution in low-calcium Gamble’s did not show a significant influence of the pre-dispersion medium , and the initial dissolution rates were comparable  and PSF  as a pre-dispersion medium. The ZnO (NM-110) concentrations after pre-dispersion in PSF showed large standard deviations throughout the testing. The measured concentrations exceeded the nominal dose of 102.4 mg/mL for all three pre-dispersion media. Inhomogeneous particle dispersions and/or variability in the dosing could explain this observation. Homogeneous suspensions and pre-dispersion stability are critical for accurate dosing [2+ ions increases with lower pH [The pre-dispersion medium affected the dissolution of ZnO (NM-110) in PSF B. No sigmparable . Howeverlower pH . The extp-value: <0.0001) and water , while there was no difference between low-calcium Gamble’s solution and the water dispersion .The coated ZnO (NM-111) demonstrated an expected lower solubility in low-calcium Gamble’s solution than in PSF A,B due tp-values in 0 + 25 min) of the 0.05% BSA dispersion was potentially influenced by particle–protein interaction as 88.3 ± 27.1 µg BSA/mg ZnO (NM-111) was found adsorbing to the surface (Despite the organic coating on ZnO (NM-111), the dissolution in PSF occurred immediately (pre-dispersion in water or PSF) or within the first hour (pre-dispersion with 0.05% BSA) B; theref surface . In all 3+ and Zn2+ may also adsorb or bind to BSA [3+ and Zn2+ after dispersion with 0.05% BSA.The observed initial suppression of Al-oxide and Zn dissolution using 0.05% BSA may be due to different mechanisms. We identified that a large amount of the BSA in the 0.05% BSA adsorb to the test materials . Howeverd to BSA , suppresd to BSA . We descScenario 1: Studies have reported that albumin can form mono and polynuclear complexes with metal ions as Zn2+, Cu2+, Pt2+, Cu2+, Ni2+, and Co2+ [3+ or Zn2+ to BSA could potentially happen on the surface of the particles as well as with ions released from the surface, forming complexes in the solution. In the case of TiO2 (NM-104), based on the assumption that all 6 wt.% Al2O3 coating is completely released from the material, aluminum will be in great excess (105 fold by molarity) compared to the BSA. One could expect that complexation could lower the concentration of free and measurable ions. The size of BSA has been reported as 66 kDa [2O3  compared to 0.05% BSA dispersions, which supports the assumption of the BSA binding to the surface of the TiO2 (NM-104) particles. Vergaro et al. investigated the interaction between nanosized TiO2 and human serum albumin and found that serum albumin interacts with TiO2 nanocrystals [crystals .3+ and Zn2+ dissolution is suppressed after dispersion with 0.05% BSA  in low-calcium Gamble’s solution and PSF  . Pre-disIn general, the use of proteins as a stabilization agent of the particle dispersion reflects the conditions in biological systems better. Further, it is an advantage of the observed delayed dissolution that it actually allows better measurement of the initial dissolution rate. While BSA is a relevant biological protein, the observation raises a question about whether a generic dispersion protocol with proteins is the best option to perform dissolution studies or dispersion should be based on a case-by-case decision. The advantage of a generic protocol is comparability between materials, repetitions, and laboratories. The choice of pre-dispersion medium may be steered by relevance for whether a biological or non-biological compartment is tested. However, a generic protocol can reach a limit, as not all NMs may be adequately dispersed using the same liquid dispersion medium. As seen in this study, the choice of dispersion medium potentially can affect the starting concentration and dissolution rates. Therefore, harmonization is required to reach good comparability between experiments and laboratories. Well-justified modifications could be accepted case-by-case. The protocol can also be improved in the future by adding measurements of initial particle concentrations, which would enhance the ability for quality control and increase the comparability of individual experiments.2 (NM-104) and ZnO (NM-111) in low-calcium Gamble’s solution and PFS and ZnO (NM-110) in low-calcium Gamble’s solution over 24 h of testing. The effect of using different pre-dispersion media shows that the choice of pre-dispersion media should be harmonized to allow the generation of comparable results between tests and across laboratories.This study demonstrated that pre-dispersion is an important parameter to consider in batch reactor dissolution testing of NMs. We showed that the choice of the pre-dispersion medium could significantly affect the dissolution profiles of TiO


Staphylococcus pseudintermedius has zoonotic potential. This pathogen exhibits multidrug resistance, including resistance to methicillin, and biofilm-forming ability, making it hard to eradicate with antimicrobial agents. One potential alternative is bacteriophage therapy. In this study, we first characterized the antimicrobial resistance profile of S. pseudintermedius from canine samples and isolated two novel bacteriophages, pSp-J and pSp-S, from canine pet parks in South Korea to potentially control S. pseudintermedius. The biological characteristics of phages were assessed, and the phages could infect most of the methicillin-resistant S. pseudintermedius strains. We found that these phages were stable under the typical environment of the body . We also assessed bacterial lysis kinetics using the two phages and their cocktail, and found that the phages could prevent biofilm formation at low doses and could degrade biofilm at high doses. Taken together, this study demonstrates that bacteriophages pSp-J and pSp-S isolated in this study can be used to potentially treat methicillin-resistant S. pseudintermedius.As a primary bacterial pathogen in companion animals,  Staphylococcus pseudintermedius is an opportunistic pathogen commonly isolated from dogs and other companion animals. Given the recent rise in pet ownership, the number of reported cases of S. pseudintermedius infection in humans has risen as well (S. pseudintermedius (MRSP) has emerged, thereby raising concerns for both veterinary and human health  specifically infect bacterial hosts and inhibit host growth. Lytic phages are major candidates as alternatives to antibiotics since they spread only by lysing the host bacterial cells. Theoretically, even just a single particle of a phage can degrade biofilms because its progeny will infect adjacent bacterial cells. Recently, phages have shown the ability to degrade the extracellular matrix of biofilms –14. Somebiofilms –17. A nubiofilms –20. Somebiofilms .S. pseudintermedius in South Korea. And then, we isolated two novel phages, pSp-J and pSp-S, from soil and water samples from pet parks in South Korea for possible application in controlling MRSP. We characterized these phages and assessed their stability in different environmental conditions. We also determined their anti-bacterial and anti-biofilm activities. We found that these phages, pSp-J and pSp-S, can prevent biofilm formation at low doses and degrade biofilm at high doses, suggesting that these phages may potentially be used for treatment of MRSP infection.In this study, we first assessed the prevalence of antimicrobial-resistant The procedures involving animals were carried out under the informed consent of the owner. To ensure the well-being of involved animals, the procedures were carefully performed by a veterinarian. The animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU-180718-3).S. pseudintermedius species specific primers  with shaking or sub-cultured on tryptic soy agar (Becton Dickinson) at 37°C. All bacterial strains used in this study are listed in Samples were swabbed from the external auditory meatus, anus, skin, and oral cavity of 208 infected dogs that visited the local veterinary hospitals in Seoul and Gyeonggi-do Korea in 2018 and 2019. The swabs were streaked on sheep blood agar  and incubated at 37°C for 1 day. Based on morphology, the presumptive staphylococci-like colonies were selected and identified using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) . The ide primers . StaphylS. pseudintermedius was prepared in Muller–Hinton broth (Becton Dickinson) and was added to sodium chloride solution  to achieve 0.5 McFarland standard turbidity. Then, the bacterial solution was inoculated by spread plating on Muller–Hinton agar using a sterile cotton swab. After allowing the inoculum to dry, the discs containing antimicrobials were placed onto the agar, and the cultures were incubated for 18–24 h at 37°C. A reference strain, Staphylococcus aureus (ATCC 25923), was used as control.The antimicrobial susceptibility profiles of the isolates were determined with disk diffusion method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines [24]. Antimicrobial agents that are generally used in veterinary hospitals in Korea were purchased from Oxoid and tested: penicillin (10 U), oxacillin (30 μg), cefazolin (30 μg), cefoxitin (30 μg), cefotaxime (30 μg), vancomycin (30 μg), gentamicin (10 μg), amikacin (30 μg), quinupristin-dalfopristin (15 μg), rifampin (5 μg), chloramphenicol (30 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), ciprofloxacin (5 μg), minocycline (30 μg), tetracycline (30 μg), linezolid (30 μg), and erythromycin (15 μg). Shortly, an overnight culture of S. pseudintermedius, we collected 150 water samples and 50 soil samples all over the South Korea for 5 months and used the MRSP strain (C21-2-1) to screen for the phages. Previously grown host strain was inoculated into a 1:1 mixture of the collected sample and TSB, followed by incubation for 24 h at 37°C. After enrichment, 10 μL of 10-fold serial dilutions (10−1 to 10−8) of culture broth were spotted on the bottom agar layered with the host strain. Samples that resulted in inhibition zones were collected by centrifugation and membrane filtration to confirm the presence of plaques using the double-layer agar method. After overnight incubation at 37°C, the plaque was cloned five times by sub-culturing a single plaque. Two phages with small and clear plaque were isolated from the different samples, designated pSp_SNUABM-J (pSp-J) and pSp_SNUABM-S (pSp-S), and used for further study.For isolation of phages that infect 4). Then, 10 μL of the phage solution (>1010 PFU/mL) was loaded on a copper grid, and the excess solution was removed using a filter paper. The phages were stained with 2% uranyl acetate for 1 min and washed three times with distilled water. The grid was air-dried, and the image was obtained with a Talos L120C transmission electron microscope  operated at 120 kV. The dimensions of the phages were calculated by measuring five independent virions.For transmission electron microscopy analysis, the phages were precipitated with polyethylene glycol-NaCl and re-suspended in SM buffer , including the 29 strains isolated in this study and 25 strains reported in a previous study (Table 1) (7 PFU/mL) was inoculated into TSA plate overlaid with each bacterial strain mixed with top agar. The plates were incubated for 18–24 h at 37°C, and the lytic ability of the phages were evaluated by the clarity of the lysis zone, which was assessed as: clear (C), turbid (T), or no lysis  was spotted on the host strain-overlaid TSA plate. The EOP values were calculated as the ratio of the average PFUs of a susceptible strain to the reference strain (C21-2-1) with three replicates.The host ranges of pSp-J and pSp-S were determined by spot assay against 54 strains of Table 1) . A drop 8 CFU/mL) was infected with the phage solution at an MOI of 0.001. Samples were taken at 0, 1, 3, 5, 10, 15, and 20 min post-infection and promptly diluted in 0.1% peptone water, followed by centrifugation at 4°C. The concentrations of the free phages were determined using the supernatants. The growth curves of pSp-J and -S were constructed by inoculating the phage solution to host strain cultures at the log phase. The phages pSp-J and -S were allowed to adsorb to the host for 5 and 10 min, respectively, followed by centrifugation. The supernatant was replaced with preheated TSB and incubated with shaking (150 rpm). Aliquots were taken every 10 min for 60 min, and the concentration of the phages at each time point was determined by the double-layer agar method. The concentration measurements were performed with three replicates.The adsorption assay was carried out as previously described . Briefly7 PFU/mL) was incubated at 4 (control), 25, 37, and 50°C. For pH stability tests, 10 μL phage suspension (1.3 × 109 PFU/mL) was used to inoculate 990 μL universalbuffer  adjusted to pH 3.0, 5.0, 7.0 (control), 9.0, and 11.0 with either 1 M NaOH or 1 M HCl . The broth was cultured with shaking, and the optical density at 600 nm (OD600) was measured every 30 min for 20 h. All tests were performed with three replicates.To evaluate the bacterial lysis effect of pSp-J, pSp-S and the cocktail (a 1:1 mixture of pSp-J and -S), host-phage co-culture was performed as previously described . Briefly8 CFU/mL) broth was inoculated into fresh broth containing 1% D-glucose and was supplemented with the stock phage solution at different concentrations . Then, 200 μL of aliquots were distributed into each well of the 96-well-plate and cultured at 37°C for 24 h without shaking. The supernatant was removed from the well, and the well was washed two times with PBS for removal of the remaining planktonic bacterial cells. Quantification of total biomass and the viable bacterial cell count in the biofilms was performed using standard plate count as previously described (n = 4). For the enumeration of viable bacterial cells in the biofilm, the biofilms formed at the bottom of the plate were scraped and re-suspended in PBS. The serial dilutions of the solution were then directly spread and cultured on the TSA at 37°C for 24 h.To evaluate the biofilm prevention efficacy of the two phages, the host bacterial strain was co-cultured with the phages in 96-well-polystyrene plates . First, 1% of an overnight bacterial culture (10escribed . The tot8 CFU/mL) was inoculated into fresh broth containing 1% D-glucose and cultured on 96-well-polystyrene plates for 24 h (n = 4). The biofilms were washed to remove the residual planktonic bacterial cells, and the phage solution with different concentrations  were treated for 24 h. Then, the supernatant was removed from the well and washed twice. The total biomass and viable cell count of the biofilm were quantified as mentioned in section Biofilm prevention assay.To evaluate the biofilm degradation efficacy of two phages, 1% of an overnight bacterial culture  using Syto 9 green fluorescent nucleic acid stain  as previously described . The bio0 formula which is recommended settings for nucleotide sequences of prokaryotic viruses  with the Bonferroni test on the SigmaPlot software . The value of S. pseudintermedius strains were newly isolated and 24 strains were from a previous study  in prevent total biomass of the biofilm . The host cell viability in the biofilms was effectively reduced by lower concentrations for pSp-J, pSp-S, and phage cocktail  were co-cultured with the host bacteria for 24 h without shaking, and changes in the degree of staining with crystal violet and changes in CFU were measured for total biomass and viable bacterial load, respectively. As shown in  biofilm  at theircocktail . The biococktail . The pSpS. pseudintermedius biofilm for 24 h without shaking. The changes in the intensity of the crystal violet stain and in CFU were measured for total biomass and viable bacterial load, respectively. The total biomass of the biofilm decreased in a concentration-dependent manner and significantly decreased at 103-109 PFU/ml of pSp-J (P < 0.05), at 104-109 PFU/mL of pSp-S (P < 0.05), and at 104-109 PFU/ml of phage cocktail  were used to treat 24 h-old Staphylococcus phage 187  which is a distinct member of the family Phietavirus, a well-known bacteriophage genus included in the Siphoviridae, infecting Staphylococcus spp. The phylogenetic analysis also showed that the phages isolated in this study constructed cluster with several unclassified siphoviruses and phage 187 in the Phietavirus consist of several subgroups rather than any other siphoviruses infecting Staphylococcus such as Biseptimavirus, Fibralongavirus, and Triavirus, which is a morphologically similar genus with Phietavirus  were obtained for pSp-J with average genome coverage of 380. The complete genome of pSp-J  contained 74 ORFs which are majority found on the positive strand (66 ORFs). A total of 10,383,812 reads  were obtained for pSp-S with average genome coverage of 381. The complete genome of pSp-S  contained 72 ORFs which also are majorly found on the positive strand (64 ORFs). The tRNA, antimicrobial resistance-, or virulence- related genes was not detected in both phages. The predicted function of the ORFs was classified into the following four categories: nucleotide metabolism , structure and packaging , lysis , and unknown function . The ORFetavirus .Totally, five colonies of original host and the re-grown bacteria after the treatment of each phages were analyzed. All the assay with chromosomal DNA showed negative for major capsid protein of pSp-J, and pSp-S. We could observe the major capsid protein band (~378 bp) only in the extrachromosomal DNA from the re-grown colonies.S. pseudintermedius has been reported in human cases  from previous study and newly isolated 29 canine strains (isolated in 2018 and 2019) from this study. The isolated bacteria were highly resistant to methicillin (82.7%), whereas previous studies have reported 40–70% MRSP isolation rates in Korea  strains and 16-28% of the tested MSSP (n = 43) strains, depending on the phages  , 48. In For biofilm-associated infection, antibiotics are not the perfect choice since biofilm formation correlates with high antibiotic tolerance . PreviouAlthough the phages have been suggested as potential alternatives to antibiotics, bacterial resistance to phages is a well-known phenomenon. The application of different phages in a combination, or a cocktail of phages, can be used to overcome this. Phage cocktails bypass the resistance to a single phage, broaden the treatment choice against multiple pathogens, and take advantage of the phages' synergistic effects –62. Howe−11 PFU, a large number of phages used for the biocontrol agent may increase the likelihood of generalized transduction (One of the major concerns on phage therapy is the possibility of transmission the antimicrobial-resistant- or virulence-related genes , 64. Howsduction . In addiS. pseudintermedius, and we were able to characterize these phages for application on biofilm prevention and degradation. Our results revealed the potential of these bacteriophages and their cocktail as alternatives to antibiotics in treating S. pseudintermedius. Further studies on the mechanism behind the poor bacterial growth inhibition capacities of high concentrations of phages can be performed to help us understand and maximize the application of these phages in treating S. pseudintermedius.Overall, we have successfully isolated two bacteriophages, pSp-J and pSp-S, that infect All datasets generated for this study are included in the article/SGK and SP contributed conceptualization and design of the study. SGK, SWK, SH, and WO conducted the experiments. SGK, SG, SY, SL, and JK analyzed and validate the data. SGK, YP, and SP supervised and reviewed the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The objective of this paper was to estimate soil moisture in pepper crops with drip irrigation in a semi-arid area in the center of Tunisia using synthetic aperture radar (SAR) data. Within this context, the sensitivity of L-band (ALOS-2) in horizontal-horizontal (HH) and horizontal-vertical (HV) polarizations and C-band (Sentinel-1) data in vertical-vertical (VV) and vertical-horizontal (VH) polarizations is examined as a function of soil moisture and vegetation properties using statistical correlations. SAR signals scattered by pepper-covered fields are simulated with a modified version of the water cloud model using L-HH and C-VV data. In spatially heterogeneous soil moisture cases, the total backscattering is the sum of the bare soil contribution weighted by the proportion of bare soil (one-cover fraction) and the vegetation fraction cover contribution. The vegetation fraction contribution is calculated as the volume scattering contribution of the vegetation and underlying soil components attenuated by the vegetation cover. The underlying soil is divided into irrigated and non-irrigated parts owing to the presence of drip irrigation, thus generating different levels of moisture underneath vegetation. Based on signal sensitivity results, the potential of L-HH data to retrieve soil moisture is demonstrated. L-HV data exhibit a higher potential to retrieve vegetation properties regarding a lower potential for soil moisture estimation. After calibration and validation of the proposed model, various simulations are performed to assess the model behavior patterns under different conditions of soil moisture and pepper biophysical properties. The results highlight the potential of the proposed model to simulate a radar signal over heterogeneous soil moisture fields using L-HH and C-VV data. Over the last few decades, the agricultural sector water demand has increased to ensure food security for a growing population ,2. In arRemote sensing has demonstrated a high potential to retrieve soil moisture from agricultural field scales to regional scales owing to the resolution and repetition frequency of remote-sensing data acquisitions compared to punctual measurements of the soil moisture ,10,11,12To relate radar signals to in situ measurements, such as soil moisture and roughness  and biopThe simplicity of the WCM makes it widely used in the literature where backscattering is the sum of the contributions of three components, namely, vegetation, soil scattering attenuated with the vegetation effect and soil-vegetation interactions. Bare soil backscattering is calculated through a wide range of models coupled to the WCM including physical approaches, such as the geometric optic model (GOM) , integraNDVI) [In previous studies relying on the WCM, the soil–vegetation interaction term has generally not been considered. However, some studies have proposed different expressions for this term, e.g., as a function of the Fresnel reflectivity and volume scattering coefficients in ,37 or viNDVI) ,44,49,59To estimate the soil moisture within a homogenous vegetation context, several techniques have been employed to invert models, including direct inversion ,49, chanWCM parametrization of vegetation and soil variables has been performed based on experimental data relating the model performance to the quality and robustness of in situ data . The sciHe et al.  proposedMost studies have been developed to retrieve soil moisture using C- and X-band SAR data rather than L-band data due to the limited availability of SAR data at the L-band frequency. The Japan Aerospace Exploration Agency (JAXA) provides Advanced Land Observing Satellite (ALOS) and ALOS-2 L-band images on request. In the near future, the European Space Agency (ESA) will deploy the Radar Observing System for Europe–L-band (ROSE-L) Mission for land and geohazard monitoring, where soil moisture retrieval is one of the main objectives . TherefoRegarding covered fields, Zribi et al.  exploredReferring to the previously mentioned works, the L-band data potential for soil moisture estimation should be explored within other contexts and assumptions. For example, radar signal modeling across spatially heterogeneous agricultural fields remains a challenge, as most approaches have been developed for homogeneous crop fields, such as cereal fields. Furthermore, localized irrigation is a source of complexity in SAR signal modeling and subsequent soil moisture estimation. In the case of drip irrigation, soil moisture is not distributed homogeneously across the entire plot . Hence, this work aims to compare the L-band and the C-band potentials for soil moisture retrieval under the same conditions, i.e., locally irrigated heterogeneous crops using a modified version of the water cloud model. This article is divided into five sections. The present study was carried out in a semi-arid area: the Merguellil Plain  in the Kairouan Plain, central Tunisia, as shown in In the study area, pepper is a leading seasonal garden crop in the summer. For three months starting from June, reference fields soil is ploughed and fertilized to prepare seed beds. After transplanting, pepper seedlings were drip irrigated. The drip irrigation is supplied by lines of irrigation tape and spaced emitters. Subsequently, leaves start to develop until inflorescence emergence and flowering. Afterwards, the flowers transform to fruits. The mature peppers are harvested multiple times until the senescence of pepper plants. (a)Optical data: Sentinel-2https://www.theia-land.fr/, accessed on 8 January 2022), already orthorectified and atmospherically corrected with a mask of clouds and shadows owing to the MAJA algorithm. After the launch of Sentinel-2 (S-2) A and B on 23 June 2015 and 7 March 2017, respectively, optical data became free and open access with a spatial resolution varying between 10 m ×10 m and 60 m × 60 m, and a revisit time of up to 5 days in 13 spectral bands at visible and mid-infrared wavelengths. In the present study, we used S-2 surface reflectance products downloaded from the Theia site SAR dataSentinel-1Eight C-band SAR images were acquired by Sentinel-1 A (S-1 A) and B (S-1 B) constellation between June and August 2020 over the study area, as described in ALOS-2NDVI images calculated from Sentinel-2 optical data with a root mean square (RMS) control point error smaller than 0.5 pixels [Advanced Land Observing Satellite-2 (ALOS-2) images are acquired at the L-band frequency by the Phased Array Synthetic Aperture Radar (PALSAR). According to 5 pixels ,49. During S-1 and ALOS-2 acquisitions (between June and August 2020), data gathering campaigns were conducted in seven irrigated reference pepper fields in the Sidi Ali Ben Salem area in the Kairouan Plain. Reference fields were considered to measure soil surface properties (roughness and soil moisture) and vegetation parameters . The surface area of these fields varied between 1 and 2.6 ha. As shown in (a)Soil moisture (Mv)In the row crop case with drip irrigation, the soil moisture spatial pattern is heterogeneous: the water content in the soil below vegetation is different from that in the bare soil part. As a result, soil moisture measurements were performed at a depth of 5 cm and with a hand-held Theta probe in each test field at 10 points randomly chosen in the bare soil part and 10 points in the soil part below vegetation. The moisture measurements were completed within two hours separating the measurements and the satellite overpass time.Within this context, we obtained soil moisture measurements collected over bare soils, namely, inter-row soil moisture For each reference field, we calculated an average soil moisture value based on the hypothesis that bare soil, characterized by According to the ground truth observations, we estimated that 15% of the field area is covered by irrigation tapes and emitters. The rest of the area, which is equal to 85%, receives irrigation water through water transfers in the soil. As a result, the measured soil moisture is the result of the contribution of soil moisture next to the emitters and 85% of the soil moisture off the irrigation zone. Therefore, in the present study, we consider that (b)Soil roughnessThe soil roughness was characterized with a 1 m pin profiler exhibiting a pin spacing of 2 cm using six roughness profiles . These profiles were digitized to calculate statistical parameters of the soil roughness, namely, the root mean square of the height (Hrms) and correlation length (Lc), as mentioned in ,49. Hrms(c)Vegetation height (H)During SAR data acquisition, as indicated in (d)Fc) and Leaf area index (LAI)Vegetation cover fraction  [In various studies, the vegetation cover fraction has been calculated as a function of the el (DPM) . In the el (DPM) ,49,74.Fc) value fluctuated across the various growth stages between 0.1 and 0.5, covering the four pepper phenological stages starting with vegetative growth at the end of June, followed by flowering, fruit setting and fruit formation. Fc and H measured over a pepper reference field where harvest event dates were different to the chosen fields in The LAI value varied from 0.07 to 1.75. The pepper cover fraction . Consequently, the vegetation contribution in the WCM was delineated by the cover fraction.The . Hence, ,47 introFc), and the other contribution Fc):In the study case involving the pepper field organization, we considered a combination of two contributions. The first contribution 2 per one m2  and (13).Linear correlation coefficient In this section, we evaluate the influence of soil moisture on the L- and C-band radar signals at the different pepper growth stages, where the pepper height value varies between 0.17 and 0.64 m, the LAI value fluctuates between 0.07 and 1.75 and Fc is less than 0.5. The objective is to conduct a preliminary analysis to detect possible soil moisture effects in high-heterogeneity context of soil moisture and vegetation cover. The initial results confirm the sensitivity of the L-band data to the average soil moisture. The sensitivity of the radar signals to soil moisture is approximately identical between the HH and HV polarizations, at 0.16 dB/vol.% and 0.18 dB/vol.%, respectively. However, the correlation is much stronger in the case of HH data. This can be explained by the volume scattering effect of the vegetation cover, which is much greater in the case of cross-polarization.Regarding the LAI parameter, radar sensitivity increases from 2.11 dB.m²/m² to 3.08 dB.m²/m² using L-HH and L-HV, respectively. C-band sensitivity increases from 0.86 dB.m²/m² in VV polarization to 2.42 dB.m²/m² in VH polarization. We also observe some sensitivity in the L-HH, L-HV, C-VV and C-VH configurations but with low R values of 0.55, 0.51, 0.39 and 0.68, respectively. This could be explained by the fact that LAI information is mainly related to the upper canopy layer and corresponds to a mean value comprehensively without reflecting the heterogenous spatial vegetation cover. The vegetation height information reflects the canopy structure the most. Therefore, the L-band exhibits a higher sensitivity to this parameter  thanks to its greater wave penetration. The C-band sensitivities to vegetation height vary between 2.85 dB/m and 5.97 dB/m for VV and VH polarizations, respectively. Within all contexts, a higher sensitivity to vegetation of the cross-polarization signals in the L-band or C-band is observed. This is related to their notable response to vegetation volume scattering. The abovementioned results emphasize the higher sensitivity of the SAR data in the cross-polarization configuration than that in the co-polarization configuration (L-HH and C-VV), as mentioned in .The comparison between the L- and C-band behaviors reveals that the radar signals increase with increasing pepper development, as manifested by the increase of radar signal as function of H and LAI. Macelloni et al.  exanimatThe same behavior was observed in previous studies using C-band ,49,86 anIn this section, we simulate the radar signal behavior in pepper fields through calibration and validation of the proposed model, as expressed in Equations (4) and (5). Based on the radar sensitivity results in Modified WCM calibration and validation were carried out through the threefold cross-validation method due to the limited data samples (30 samples for L-band data and 32 samples for C-band data). This method consisted of a resampling procedure starting with random shuffling of the dataset followed by splitting into three partitions of equal size. With each partition, we calibrated the proposed model on 2/3 of the whole dataset, and we validated the model against 1/3 of the dataset. In each step, we evaluated the method performance by calculating the statistical parameters of the bias and RMSE.Hence, A and B were calibrated three times. We only retained the most accurate calibration results in the subsequent steps. The A parameter values varied between 0.27 and 0.5 and B values fluctuated between 0.5 and 2.68, for L-HH and C-VV, respectively. The best WCM calibration performance was characterized by RMSE values ranging from 0.58 dB to 1.33 dB for the C-VV and L-HH data, respectively. The slightly higher RMSE in the L band could be explained by a greater roughness effect and surely a greater sensitivity to the entire canopy structure at this frequency band. In this section, we analyze the behavior of the backscattering coefficient as function of soil moisture using the calibrated modified WCM in the case of pepper fields, considering a large range of vegetation and soil conditions: bare soil moisture ranging from 5 to 40 vol.%, underlying soil moisture ranging from 10 to 40 vol.%, pepper height ranging from 0.1 to 0.7 m, and vegetation cover fraction ranging from 0.1 to 0.6. In all simulations, we consider the hypothesis of constant roughness parameters (Hrms = 0.8 cm and Lc = 5 cm) as input into the IEM-B model to minimize the roughness effect. The principle of this analysis encompasses fixing a given condition and varying the other conditions to evaluate the slope of the linear relationships between soil moisture and modeled backscattering coefficients in the L-HH and C-VV configurations. We simulate the SAR backscattering coefficients with constant values of the cover fraction. At each value of the pepper coverage fraction, we varied the vegetation height level and recorded the slope (dB/vol.%). Fc and H values to 0 dB/vol.% at an Fc value of 0.3 and a pepper height of 0.3 m. To better investigate the model sensitivity with vegetation parameter variation, we generated 3D surface scatter plots of the modified WCM sensitivity to the different soil moisture values according to the above established linear relationships as a function of the cover fraction and vegetation height  under diTo further investigate the evolution of model sensitivities as function of vegetation height and cover fraction, we have chosen to present 2D profiles extracted from Using C-VV data, the simulation slope fluctuates between 0.17 dB/vol.% and 0 dB/vol.%. For a vegetation height equal to 0.1 m, L-HH sensitivities vary from 0.25 to 0.05 dB/vol.% regarding a slope between 0.17 and 0.03 dB/vol.% using C-VV for Fc = 0.1), the comparison between L-HH and C-VV simulation results reveals that the modified WCM, using L-HH data, remains sensitive to soil moisture as function of the increase pepper height and the inter-rows soil water content. However, the proposed model using C-VV becomes insensitive to soil moisture with the vertical development of pepper plants. This C-band behavior is induced from a strong attenuation of the signal over peppers. Pepper height reaches its maximum at 0.7 m. For this case, L-HH signal slopes vary from 0.05 dB/vol.% to 0.01 dB/vol.% regarding zero values for C-VV simulations. With the increase of pepper’s height, the dynamic of model sensitivities, using L-HH data, decreases from 0.20 dB/vol.% to 0.04 dB/vol.% for H values equal to 0.1 m and 0.7 m, respectively. We notice low sensibility characterizing C-VV simulations where sensitivity decrease from 0.14 dB/vol.% to be null, for vegetation heights equal to 0.1 m and 0.7 m, respectively. Under low vegetation cover fraction  and horizontally (cover fraction) and inter-row soil moisture values. For constant values of cover fraction values, the dynamics of the sensitivity to soil moisture decreases as function of the increase of pepper height. This decrease may indicate the domination of bare soil the total backscattering mechanism until the pepper height reaches the value of 0.3 m for the C-VV signal and 0.5 m for the L-HH signal. Subsequently, vegetation backscattering contribution increases with the pepper growth regarding the decrease of bare soil impact. The comparison of simulation results underlines that model sensitivity based on C-VV data decreases more rapidly than does that based on L-HH data as a function of the pepper cover fraction and height and the soil water content. The L-HH data sensitivity declines and remains relatively higher than the C-VV data sensitivity given the same values of Fc and H. The aforementioned results also highlight the potential of the modified WCM to simulate SAR signals in the pepper reference fields. Conversely, the proposed model potential should be further explored considering other heterogeneous plants under drip irrigation via a comparison to the present study.This study proposes an analysis of the potential of SAR data for soil moisture retrieval in drip-irrigated pepper fields in semiarid areas at the center of Tunisia, i.e., the Kairouan Plain, using L- and C-band SAR data. By considering that 15% of the field area is influenced by drip irrigation and that the remaining area is dominated by non-irrigated bare soil, we analyzed the sensitivity of radar signal to the weighted soil moisture content . Using L-HH data, the proposed model remained sensitive to soil moisture where sensitivity values vary between 0.25 dB/vol.% and 0.08 dB/vol.% at a vegetation height and a cover fraction values less than 0.5 m and 0.4, respectively, where inter-row soil moisture value is equal to 5 vol.%. Under the same soil moisture context, the model sensitivity, using C-VV data, decreased from 0.17 dB/vol.% to 0.08 dB/vol.% at a vegetation height less than 0.3 m and a cover fraction value lower than 0.3. The aforementioned vegetation biophysical parameters thresholds vary as function of the inter-row vegetation soil moisture contexts. One of the limitations of the proposed semi-empirical approach is its dependence on the database used and the study objectives. Hence, the suggested approach should be tested at other sites. The inversion process of the modified WCM coupled with the IEM-B model for soil moisture retrieval purposes is complex to realize. Therefore, machine-learning algorithms, such as artificial neural networks (ANNs), will be applied in future work to estimate the soil water content.


The present study explored the predictors of the development of liver-related events in HCV cirrhotic subjects achieving SVR following antiviral therapy with direct-acting antiviral agents (DAAs) during a follow-up of 24 months after SVR confirmation. Patients had a liver stiffness measurement (LSM) of ≥14 kPa at baseline. We found that baseline liver stiffness ≥ 20 kPa and HCV genotype different from 1 were both independent predictors of liver decompensation, while only LSM ≥ 20 kPa was an independent predictor of HCC.p < 0.03). The incidence rate x 100 p/y was 1.6 for HCC, 1.7 for any liver decompensation, and 0.5 for hepatic death. Baseline liver stiffness (LSM) ≥ 20 kPa  and genotype different from 1  were both independent predictors of liver decompensation. Baseline LSM > 20 KPa  was the sole independent predictor of HCC. A decrease in liver stiffness (Delta LSM) by at least 20% at the end of follow-up was not associated with a decreased risk of liver-related events. Conclusion: Baseline LSM ≥ 20 kPa identifies HCV cirrhotic subjects at higher risk of liver-related events after SVR.Background: Prospective studies on predictors of liver-related events in cirrhotic subjects achieving SVR after DAAs are lacking. Methods: We prospectively enrolled HCV cirrhotic patients in four Italian centers between November 2015 and October 2017. SVR and no-SVR cases were compared according to the presence or absence of liver-related events during a 24-month follow-up. Independent predictors of liver-related events were evaluated by Cox regression analysis. Results: A total of 706 subjects started DAAs therapy. SVR was confirmed in 687 (97.3%). A total of 61 subjects (8.9%) in the SVR group and 5 (26.3%) in the no-SVR group had liver-related events ( Experience with new direct-acting antiviral agents (DAAs) treatment for chronic HCV infection has shown that more than 95% of treated subjects, even those with liver cirrhosis, achieves sustained virological response (SVR), i.e., clearance of the virus . This raSeveral studies compared SVR versus no-SVR cases to assess the rate of independent predictors for liver-related events. Cirrhotic subjects who achieved SVR after DAAs treatment had significantly lower rates of liver-related events, including de novo HCC development, as compared to those who did not achieve SVR ,5,6,7,8.Nonetheless, cirrhotic subjects who achieved SVR continued to develop liver-related events, albeit at a lower rate . There iTwo US retrospective studies ,11 and oTo further expand the currently limited information on this issue, we evaluated the non-serological baseline predictors of liver-related events’ development in cirrhotic subjects (regardless of Child–Pugh stage) achieving SVR after DAAs therapy in a prospective manner.p < 0.003 by log-rank test).A total of 706 HCV-RNA-positive cirrhotic patients started DAAs therapy. SVR was confirmed in 687 (97.3%) of them. A total of 66 subjects had at least one liver-related event during a mean follow-up of 28.4 ± 5 months: 61 (8.9%) in the SVR group and 5 (26.3%) in the no-SVR group. Ten subjects  were lost to follow-up; none of them, however, reported liver-related events up to the last observation performed. The Kaplan–Meier cumulative incidence curves of any event by SVR status are shown in The incidence rate of any event  per 100 person-years was 3.7 (95% CI 2.9–4.8) in the SVR group and 12.3 (95% CI 5.1–29.6) in the no-SVR group. The relative rate (RR) of any event for subjects who did not achieve SVR compared to subjects who achieved SVR was 3.3 (95% CI 1.3–8.2).In SVR patients, eight liver-related deaths were recorded, and two were recorded in no-SVR patients. The incidence rate per 100 person-years was 0.5 (95% CI 0.2–1.0) in the SVR group and 4.3 (95% CI 1.1–17.2) in the no-SVR group. The RR or incidence rate ratio for subjects who did not achieve SVR compared to subjects who achieved SVR was 9.0 (95% CI 1.9–42.6). See The baseline characteristics of the 687 subjects who achieved SVR are shown in In total, 15.4% (106/687) of SVR subjects had an improvement in the Child–Pugh score at the last evaluation in comparison to the baseline value. In particular, 72.7% of Child–Pugh B patients had an improvement in liver function, obtaining a Child–Pugh score of A after SVR.The incidence rate × 100 person-years was 1.2 for overall death (0.5 for hepatic and 0.7 for not-hepatic cause), 1.7 for liver decompensation, 1.6 for HCC, and 0.1 for liver transplantation. The overall incidence of any event was 3.7 × 100 person-years .p < 0.04)  of the 567 subjects who had a liver stiffness measurement at the beginning of the study. Of them, 258 (52.6%) had a ≥20% decrease in liver stiffness value as compared to baseline. The only baseline factor associated with a liver stiffness decrease of ≥20% was a genotype different from 1 ( < 0.04) . CompariThe crude and adjusted HR for the association of non-serological baseline characteristics of SVR subjects with liver-related events are shown in Until now, the largest published cohort of cirrhotic patients treated with DAAs outside a liver transplant setting is the study reported by Cheung et al. , which aThe main finding of the present study is the role of baseline liver stiffness (≥20 kPa) as an independent predictor of liver-related event development. Compared to subjects with baseline liver stiffness <20 kPa, those who had an LSM above this cut-off showed a four-fold higher risk of liver decompensation and a seven-fold higher risk of HCC after adjustment for the confounding effect of baseline variables by the Cox regression model.Liver stiffness measurement by transient elastography is an accurate indirect, non-invasive method to assess liver fibrosis grade , a stronAn increased basal LSM value, evaluated as a continuous variable, has been associated with an increased risk of HCC in several studies ,24,25,26The cut-off of 20 kPa identified in the present study is in line with the data produced by Lens et al., who identified a cut-off of 21 kPa to separate patients with and without benefit from SVR in the presence of clinically significant portal hypertension .In agreement with our findings, a previous retrospective Italian study  using a Change in liver stiffness value over time (Delta LMS) has been proposed as an accurate and reliable predictor of liver decompensation  and HCC Compared to Child–Pugh A cases, the enrolled Child–Pugh B cases did not show a higher risk of subsequent liver decompensation or HCC. Additionally, most subjects showed an improvement in liver function, becoming Child–Pugh A with a subsequent decreased risk of liver-related events. Conversely, a recent retrospective study has shown that in patients with chronic HCV infection, DAAs-induced SVR was associated with a reduced risk of liver-related events in Child–Pugh A subjects but not in those with Child–Pugh B/C cirrhosis . These dOur study shows low incidence rates × 100 p/y of liver-related events in SVR cirrhotic subjects. The incidence estimate rate of HCC is 1.6 × 100 p/y; it is similar to the 1.5 rate × 100 p/y reported in the recent prospective Spanish study , to the In our study, the incidence rate × 100 person-years of follow-up was 1.7 for liver decompensation events. In particular, 28 patients (4%) developed at least one liver decompensation event . These data are in line with those of Cheung et al., who showed that liver-related events were most frequent during the treatment period and decreased over time with 16/317 SVR patients (5%) experiencing decompensation in 6–15 months after DAA treatment [The main limitation of this study is the exclusion of Child–Pugh C patients, who, according to the Italian DAAs prescription rules, are not suitable for treatment outside the liver transplantation waiting list. As these subjects are at higher risk of liver-related events, their exclusion may have generated an underestimation of this outcome.On the other hand, the present study is characterized by several strong points that are worth underlining. First, and most importantly, its prospective design allows for more accurate ascertainment than retrospective studies, which may be affected by selection and ascertainment biases. Second, the large patient cohort, the consecutive enrollment of cases, and the long follow-up (28.4 ± 5 months) represent a unique example in the literature. Third, the cut-off of basal LSM adopted for enrolment (≥14 kPa) allows excluding patients with bridging fibrosis, a subgroup at a lower risk of liver-related events as compared to cirrhotic ones. Finally, the large proportion (86.5%) of subjects who had a coupled liver stiffness measurement at baseline and at the end of follow-up provided enough statistical power to detect the influence of Delta LSM on the occurrence of liver-related events.This was a prospective cohort study. All cirrhotic subjects receiving DAAs therapy were followed for 24 months after confirming SVR (see paragraph SVR). At the end of follow-up, outcome event rates were compared between SVR and no-SVR. Among SVR cases, a further comparison was made among those who developed and those who did not develop liver-related events. In this latter group, adjusted hazard ratios (HR) were calculated to evaluate independent non-serological baseline event predictors .All consecutive HCV-RNA positive cirrhotic subjects, ≥18 years of age, and Child–Pugh stage A and B candidates for DAAs therapy were enrolled in this prospective study between 1 November 2015 and 31 October 2017 in four tertiary centers .The diagnosis of liver cirrhosis was based on the presence of the peculiar clinical, biochemical, ultrasound signs , and traA web database recorded information on baseline demographic, clinical, and treatment characteristics of patients; treatment outcome, type, and timing of event occurrences were also registered.Subjects signed informed consent to participate. The study was conducted in accordance with the Declaration of Helsinki. The protocol was approved by the ethical board of the promoting center (Federico II University of Naples).SVR was defined as undetectable HCV-RNA at 12 weeks after the end of therapy using the Cobas AmpliPrep/Cobas TaqMan .Liver stiffness measurement (LSM) was performed using transient elastography at baseline and at the end of follow-up by a single experienced operator in each center and according to the usual standard procedure. Improvement in LSM at the end of follow-up (Delta LSM) was defined as a decrease of ≥20% from basal LSM value.Recurrence or new-onset of 1. ascites, 2. variceal bleeding, 3. spontaneous bacterial peritonitis, 4. hepatic encephalopathy for Child–Pugh A and B patients;Occurrence of de novo HCC for Child–Pugh A and B patients;In Child–Pugh B patients, worsening of a pre-existing symptom of decompensated cirrhosis .The main outcome of the study was the analysis of liver-related events development after SVR achievement. Liver-related events were defined as:Any liver-related event that developed during follow-up was registered in the database. Mortality was also registered as liver-related or not liver-related. HCC surveillance was performed with ultrasound every 6 months according to standard clinical practice. In the case of liver focal lesions identified by ultrasound, HCC was confirmed by imaging (computed tomography and/or magnetic resonance imaging) and/or biopsy examination according to international guidelines .The at-risk period for each subject was defined by the time from starting DAAs treatment until the onset of the event. Events occurring before the end of antiviral treatment were not included in the analysis . Follow-up lasted 24 months after SVR.Data were censored when individuals died or were lost during follow-up. Patients lost during follow-up were registered as alive until the last day of observation.p-value < 0.05 was considered significant.Continuous variables were expressed as mean and standard deviation (SD), and qualitative variables as absolute frequency and percentage. Differences in proportions were evaluated by the Chi-square test; a The relative rate (RR) of liver-related events was calculated as a ratio of the incidence rates of events in the overall cohort of subjects according to the SVR status and in the SVR cohort according to the baseline LSM and its decrease from baseline to the end of follow-up by at least 20%.The Kaplan–Meier method was used to estimate the cumulative incidence of events. Comparison of survival curves between groups was performed using the log-rank (Mantel–Cox) test.Incidence density rates of liver-related events per 100 person-years, with 95% confidence intervals (95% CI), were provided.p-value lower than 0.10 at univariate analysis were included in the multivariate model. A sensitivity analysis was performed using Cox proportional hazards regression accounting for the competing risk [In SVR patients, univariate and multivariate Cox regression analyses were used to identify baseline variables  associated with the development of any liver-related event or HCC (outcome variables). Variables with a threshold ing risk , consideThe association of baseline characteristics in SVR subjects with an LSM decrement by ≥20% from baseline to the end of follow-up was assessed by a logistic model.Statistical analyses were performed using STATA 14.2 statistical software .In conclusion, this study identified a basal LSM cut-off of ≥20 kPa as an independent predictor of liver-related events in cirrhotic subjects achieving SVR after DAAs therapy. Studies with a longer follow-up in this setting are warranted to further evaluate the potential role of Delta LSM on the occurrence of liver-related events.


Current pharmacological intervention for the cancer-related pain is still limited. The aim of this study was to explore whether repetitive transcranial magnetic stimulation (rTMS) could be an effective adjuvant therapy to reduce pain in patients with advanced non-small cell lung cancer (NSCLC).This was a randomized, sham–controlled study. A total of 41 advanced NSCLC patients with uncontrolled pain  were randomized to receive active  (n = 20) or sham rTMS (n = 20) for 3 weeks. Pain was the primary outcome and was assessed with the Numeric Rating Scale (NRS). Secondary outcomes were oral morphine equivalent (OME) daily dose, quality of life , and psychological distress . All outcomes were measured at baseline, 3 days, 1 week, 2 weeks, and 3 weeks.p < 0.001, Cohen’s d =1.135) and week 3 . The OME daily dose, physiology and psychology domains of WHOQOL-BREF scores, as well as the HAM-A and HAM-D scores all were significantly improved at week 3 in rTMS group.The pain intensity in both groups decreased gradually from day 3 and decreased to the lowest at the week 3, with a decrease rate of 41.09% in the rTMS group and 23.23% in the sham group. The NRS score of the rTMS group was significantly lower than that of the sham group on the week 2 (Advanced NSCL patients with cancer pain treated with rTMS showed better greater pain relief, lower dosage of opioid, and better mood states and quality of life. rTMS is expected to be a new effective adjuvant therapy for cancer pain in advanced NSCLC patients. Recently, the incidence of cancer in the world is increasing year by year . AccordiThe WHO three-step ladder for cancer pain, which is widely used in clinic, follows the rule that the choice of analgesic drugs were based on the pain intensity: step I-nonsteroidal anti-inflammatory drugs  to mild pain, step II- weak opioids  to moderate pain, and step III- strong opioids (morphine or oxycodone) to severe pain . MeanwhiIn the past two decades, neuromodulation technique has gradually become a new direction of pain treatment. Repetitive transcranial magnetic stimulation (rTMS) is one of the most commonly used non-invasive neuromodulation techniques in clinic . rTMS coIn addition, patients with cancer pain are often complicated with depression, which aggravates the complexity and difficulty of cancer pain treatment, and that is also one of the main reasons for poor analgesic effect . Many stThis trial was a randomized, double-blind, parallel-group, sham-controlled clinical trial, approved by the Ethics Committee of Chongqing Cancer Hospital and was registered (ChiCTR.org.cn identifier: ChiCTR 2000029130). All patients provided written informed consents before enrolling. After completing consent forms, patients were randomly assigned to either the rTMS group or the sham stimulation group using a block randomization scheme. An independent investigator carried out the randomization. Two independent, trained assessors and enrolled patients were blinded to the group assignments.NSCLC patients with cancer pain were enrolled consecutively from January 2020 to March 2021. The inclusion criteria were: 1) confirmed diagnosis of advanced NSCLC by pathology or cytology, 2) accompanied with pain symptoms, and confirmed as cancer pain by oncologist, 3) experienced worst pain score≥4  at the site of pain, 4) age between 18 and 70 years, 5) with clear awareness, and could cooperate to evaluate pain severity, 6) estimated that the survival time is more than 3 months, 7) with completion of signed informed consents, and voluntary participation in this study. The exclusion criteria were: 1) brain tumor patients 2) history of seizure, 3) implanted pacemaker, stent and other metal substances 4) acute pain anywhere in the body due to other diseases, 5) serious psychiatric diagnoses All the participants were treated with medications according to WHO three-step principle. In order to facilitate the statistics of the dosage of opioids in patients with cancer pain, morphine sulfate controlled-release tablets or oxycodone hydrochloride sustained-release tablets were used for analgesia in this study .rTMS was applied with a magnetic stimulator (CCY-I) with a figure-of-eight coil . The rTMS protocols used in this study were in accordance with the safety guidelines for rTMS applications . ParametThe pain intensity was assessed at 1 day before rTMS treatment (T0), 3 days (T1), 1 week (T2), 2 weeks (T3) and 3 weeks (T4) after first rTMS treatment. Mood status and quality of life were evaluated only at T0 and T4 timepoints. 1. NRS. The NRS consists of 11-point scale, of which 0 represents no pain and 10 represents the strongest pain imaginable, which has been recommended by the Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT) guidelines .The secondary outcomes were as follows:1. Oral morphine equivalent (OME). The conversion method of OME refer to the previous literature reports: oral oxycodone hydrochloride is 1:1.5, intravenous morphine is 1:3 converted to oral morphine .2. WHO Quality of Life-BREF (WHOQL-BREF) . Quality3. Hamilton Anxiety Scale (HAM-A) . HAM-A w4. Hamilton Depression Scale (HAM-D) . HAM-D wP<0.05 (two-sided test) and an effect size of r = 0.45, a total minimum sample size of 38 patients was required. Considering an estimated 10% dropout rate, the sample size was inflated to 21 participants per group (N = 42).Sample size was estimated using the G Power v.3.1 statistical tool. To achieve a statistical power of 85% with statistical significance at t tests or chi-squared tests. A repeated-measures analysis of variance (rmANOVA) was used to analyze the data for the efficacy of rTMS, with time as the within-subjects factor and treatment as the between-subjects factor. Post hoc analyses were performed with Bonferroni adjustment for further multiple comparisons. P<0.05 were considered statistically significant. The percentage of change within each individual was calculated as follows: [(post-treatment - pre-treatment score)/(pre-treatment score)] *100.Statistical data analyses were performed using IBM SPSS . Baseline group differences were explored with A total of 63 advanced NSCLC patients with cancer pain were screened and 42 eligible patients were enrolled in the study Figure 2P = 0.04, Cohen’s d =-0.735) and week 3   in the rTMS group and 6.37  in sham group. The pain intensity in both groups decreased gradually from day 3 and decreased to the lowest at the week 3, with a decrease of 41.09% in the rTMS group and 23.23% in the sham group. The NRS score for the rTMS group was significantly lower than that of the sham group on the week 2 (- 0.822) Figure 3P = 0.02, Cohen’s d = 0.796), while the sham group both were significantly higher than that of baseline   Figure 4P = 0.02, Cohen’s d = 0.796 and P = 0.031, Cohen’s d = 0.746, respectively). . Figure 4P = 0.005, Cohen’s d = -0.949 and P = 0.011, Cohen’s d =- 0.869, respectively). However, there were no significant improvements in the sham group  for the publication of any potentially identifiable images or data included in this article.YT and Y-sL conceived and designed the study. YT and HC analyzed the experimental data and drafted the manuscript. YT, M-lT, and YZ collected the experimental data. Y-sL, S-lX, YL and X-hJ revised the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version.This study was supported by the National Nature Science Foundation of China (81902302) and Chongqing Natural Science Foundation (cstc2018jcyjAX0772).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.